SI22338A - Diazenedicarboxamides as inhibitors of d-alanyl-d-alanin ligase - Google Patents

Abstract

Described are compounds of the general formula (I) and pharmaceutically acceptable salts of these compounds. Individual substituents are clearly defined in the text and claims. The compounds are effective inhibitors of the enzyme D-alanyl-D-alanin ligase (Ddl).

Description

DIAZENDICARBOXAMIDES AS D-ALANYL-D-ALANINE LIGASE INHIBITORS

Description of the invention

FIELD OF THE INVENTION

The invention relates to the field of pharmaceutical chemistry and relates to novel diazendicarboxamides as inhibitors of bacterial D-alanyl-D-alanine ligase (hereinafter Ddl), to processes for their preparation, to use and to pharmaceutical products containing them. New diazendicarboxamides are inhibitors of the enzyme Ddl, which is involved in the bacterial peptidoglycan biosynthesis and has antibacterial activity.

A technical problem

The resistance of pathogenic bacteria to the antibacterial agents used today to treat bacterial infections is increasing (Ritter, TK; Wong, C.-H. Angew. Chem. Int. Ed. 2001, 40, 3508-3533; Davies, J. Nature 1996, 383, 219-220; Levy, SB Sci.Am 1998, 278, 4653). Certain strains of three bacterial species (Enterococcus faecalis, Mycobacterium tuberculosis, Pseudomonas aeuginosa) that can cause life-threatening infectious conditions have developed resistance to all antibiotics used in therapy. Therefore, there is a need for new antimicrobial agents that will work on the unused sites in bacteria.

The state of the art

The most important building block of a bacterial cell wall, peptidoglycan, is a unique structure present only in bacteria. The early stages of peptidoglycan biosynthesis, which take place in the cytoplasm, are still relatively untapped sites and therefore suitable for the development of a new type of antimicrobial agents. The amino acid ligases MurC, MurD and MurE catalyze the attachment of L-alanine (LAla), D-glutamic acid (D-Glu) and meso-diaminopimelic acid, respectively. Of L-lysine (L-Lys) in emerging UDP-A-acetylmuramoyl-pentapeptide, whereas MurE catalyzes the attachment of the D-alanyl-D-alanine dipeptide (van Heijenoort, J. Nat. Prod. Rep. 2001, 18, 503-519 ). Two enzymes are responsible for the synthesis of this dipeptide, alanine racemase (catalyzes the conversion of L-alanine to D2 alanine) and Ddl. The enzyme inhibitors listed are potential antibacterial agents.

Ddl is a cytoplasmic enzyme that catalyzes the formation of the D-alanyl-D-alanine dipeptide according to the following reaction:

D-Ala + D-Ala + ATP <- »D-Ala-D-Ala + ADP + Pi

The acyl-phosphate reaction mechanism is similar in Ddl to that of other ΑΤΡ-dependent ligases (eg glutamine synthetase, glutathione synthetase, Mur ligases). The enzyme first phosphorylates the carboxyl group of the D-alanine residue of the nucleotide substrate with the γ-phosphate group of ATP to form an acyl-phosphate intermediate. The carboxyl group thus activated is attacked by the free amino group of the second D-alanine. This results in a tetrahedral transition state. The elimination of inorganic phosphate and peptide bond formation follows. The enzyme also requires Mg ²⁺ to function smoothly, coordinating the correct placement of the substrate in the active site, regulating charge in the active site, and subsequently stabilizing the complex between the substrate and the enzyme. The acyl-phosphate mechanism was also confirmed by the crystalline structure of the enzyme-phosphorylated phosphinate inhibitor complex (Fan, C 'Park, IS, Walsh, CT, Knox, JR Biochemistry 1997; 36: 2531-2538).

Two isomorphic forms of Ddl are known: DdlA and DdlB. Despite differences in size (the DdlB enzyme is smaller) and only 35% homology to the amino acid sequence, the enzymes have very similar catalytic efficiency and ability to identify substrates (Zavvadzke, LE, Burr, TDH, Walsh, CT: Biochemistry 1991; 30: 1673-1682) . The similarity of both enzymes is also evident in sensitivity to inhibitors.

Most important among Ddl inhibitors is D-cycloserine, which is clinically used as a tuberculostatic (Wargel, R. J., Shadur, C. A., Neuhaus, F. C.: J. Bacteriol 1970; 103: 778-788). D-cycloserine also inhibits the alanine racemase enzyme, which forms a substrate for Ddl. Many Ddl inhibitors are also known to act as transient mimetics. Several phosphate inhibitors of Ddl have been synthesized, which also confirmed the putative acyl phosphate mechanism of the enzyme. These compounds had a high inhibitory activity, as their tetrahedral geometry, after enzymatic phosphorylation, mimics the natural transition state intermediate (Chakravarty, PK, Greenlee, WJ, Parsons, WH, Patchett, AA, Combs, P., Roth, A., Busch , RD, Mellin, TN: J. Med Chem. 1989; 32: 1886-1890; Busch, RD, Mellin, TN, Valiant, ME, Weissberger, B., Gadebusch, H., Springer, JP, Combs, PL , Davidson, J., Taub, D., Schoen, W.R., Buli, H.G., Patchett, A.A., Parsons, W.H .: J. Med. Chem. 1988; 31: \ Π2- \ ΊΊ8). The years

In 1996, Bartlett and co-workers prepared phosphinate and phosphonate dipeptide analogues of the transition state, which well inhibited both DdlA and DdlB (Tom, N. J., Ellsworth, B. A., Bartlett, P. A.: Chem. & Biol. 1996; 3: 37-44). Recently, a novel cyclopropyl inhibitor Ddl (Lloyd, AJ, Roper, DI, Chopra, I., Stubbings, W., Bostock, J.M., Besong, G.E., Fishvvick was synthesized based on computer-aided de novo enzyme inhibitor design (SPROUT software) , CWG, Johnson, P.: Angew. Chem. Int. Ed. 2005; 44 · 2-6).

Description of solution to a technical problem with implementation examples

The invention relates to novel compounds of general formula (I)

X O O X

1 ^{1 11 11 1} 2 2 w — Y— NC- N = NC- NYW (i) in which:

X ¹ , X ² : hydrogen, C 1-6 linear or branched alkyl, (aryl) -NH-, -O-, - (CH 2) _n - (wherein n may be 0 to 5), C _1-6 branched alkyl;

Υ ¹ , Y ² : - (CH 2) _n - (wherein n may be 0 to 5), C 1-6 branched alkyl; -NH-, -C = O; -NHC0-,

-C0NH-;

W ', W ² : - hydrogen,

- C1-6 linear or branched alkyl which may be substituted by one or more nitro groups, by one or more halogens, by trifluoromethyl group, by one or more hydroxy groups,

- phenyl which may be unsubstituted or substituted by one or more nitro groups, by one or more halogens, by trifluoromethyl group, by one or more hydroxy groups, by one or more C1-8 linear or branched alkyl, by one or more Ci -g linear or branched alkoxy group, with one or more unsubstituted or substituted phenyl groups,

- unsubstituted or substituted naphthyl,

- fluorenyl,

- a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic ring system which may be substituted or unsubstituted and which may contain, in addition to carbon atoms, heteroatoms selected from N, O, P and S,

- a 3- to 10-membered monocyclic or 7- to 10-membered bicyclic carbocyclic ring system, which may be substituted or unsubstituted and which may contain, in addition to carbon atoms, up to 3 heteroatoms selected from N, O, P and S.

Some compounds of the present invention have one or more stereogenic centers at which the absolute configuration may be R or S and may occur in the form of racemates, conglomerates, pure enantiomers, mixtures of diastereoisomers or pure diastereoisomers.

The invention also relates to pharmaceutically acceptable salts of compounds of formula I.

The invention also relates to the use of compounds of formula I as therapeutically effective substances for the preparation of medicaments. The new compounds are inhibitors of the D-alanyl-D-alanine ligase enzyme and are used to treat infections.

The invention also provides pharmaceutical compositions containing compounds of formula I. They may take the form of various injections, oral preparations or other suitable pharmaceutical forms. In addition to the active substance, they contain various standard additives, depending on the type of use. Pharmaceutical preparations are prepared by standard procedures. They can also be formulated in various forms to ensure controlled and prolonged release of the active substance. The dose, frequency and method of administration depend on various factors and depend on the individual substance and its pharmacokinetic properties as well as the patient's condition.

The invention also relates to processes for the preparation of compounds of formula I.

Diazendicarboxamide derivatives of general formula I are prepared:

A non-symmetric derivative is formed by reacting the isocyanate of formula II (prepared from the corresponding amine with phosgene, diphosgene or triphosgene in an aprotic dry solvent in the presence of a tertiary amine) with carbazate III (R = C [ _.6 linear or branched alkyl) in an aprotic solvent.

W ¹ —Y — N — C = O

HNN — OR ² H (Π) (III)

The corresponding 1,4-disubstituted semibarbazide IV is obtained. The same compound can be prepared from

Of 4-substituted semicarbazide V when it is reacted with chloroformate VI (R = C 1-6 linear or branched alkyl).

X ¹ OOX ¹ O,, I II ¹¹ 2 2 1 1 ^{1 11} O w — YNCNNCOYW W — Υ-NCN-NH, II

HHH ² Cl-OR (IV) (V) (VI)

The resulting 1,4-disubstituted semicarbazide IV is converted with a suitable organic or inorganic oxidant into aminocarbonyl diazenecarboxylate VII. The latter is converted to diazendicarboxamide I by substitution of an alkoxy group with primary or secondary amine VIII.

χ ²

X ¹ OOI 2 ,. , I II II N — Y — Vv w — YNCN = NC-OR H (VII) (VIII)

2. The symmetric derivative of diazendicarboxamide I (X ¹ = X ² , Υ ¹ = Y ² , W '= W ² ) is obtained if the dialkyl diazendicarboxylate is treated with at least two equivalents of primary or secondary amine VIII in a protic or aprotic solvent, or may be the presence of a solvent, the nucleophilic substitution of the two alkoxy groups proceeding.

Biological tests

I. Enzyme Method for Determining the Effectiveness of DdlB Enzyme Inhibitors

1. Principle

The DdlB enzyme catalyzes the formation of dipeptide, D-alanyl-D-alanine (D-Ala-D-Ala) from D-alanine (DAla) in the presence of ATP, which decomposes into ADP and phosphate. Phosphate formation was determined spectrophotometrically at 650 nm indirectly via the formation of a green colored complex with malachite green (Biomol reagent). The catalytic activity of the DdlB enzyme is evaluated based on the amount of phosphate produced by the reaction. The amount of phosphate formed must be within the linearity range of the phosphate calibration curve. When the inhibitor is added, the catalytic activity of the DdlB enzyme is reduced, so the amount of phosphate produced is lower compared to the control without the inhibitor. The inhibitor efficiency is expressed by the residual activity (RA) of the enzyme at a given inhibitor concentration and by a constant of IC50.

2. Reagents

DdlB: Isolated and completely purified from E. coli strain JM109 (plasmid pQE-30UA). The purified enzyme is stored at - 20 ° C. Immediately before use, the enzyme is diluted with enzyme dilution buffer.

An enzyme dilution buffer of the following composition:

Hepes 20.0 mM dithiothreitol (DTT) 1.0 mM pH was adjusted to 7.2 by addition of HCl solution.

Reaction mixture (RM) for DdlB; Prepare the reaction mixture with the following composition:

Hepes (pH = 8.0)

MgCl ₂ (NH ₄ ) ₂ SO ₄

ATP

D-Ala

KC1

50.0 mM

5.0 mM

10.0 mM

769.0 μΜ 1077.0 μΜ

15.4 mM

The final concentrations of the RM constituents on the microtiter plate with the test solution are: Hepes 38.5 mM

MgCl ₂ 3.25 mM (NH ₄ ) ₂ SO ₄ 6.5 mM

KC1 10.0 mM ATP 500,0 μΜ L-Ala 700,0 μΜ

Inhibitors; prepare stock solutions of inhibitors (active substances) in DMSO at a concentration of 10 mM. If the test substances at the basic concentration are insoluble in the reaction mixture, they are diluted with DMSO 2-10 times, respectively. Also, for the determination of IC50, working solutions are prepared by diluting the stock solution with DMSO. The maximum concentration of DMSO in the test solution does not exceed 5%.

3. The procedure or. Workflow

Measurements are performed on microtiter plates. Pipette 32.5 μΐ of the reaction mixture (RM) into each well, add 2.5 μΐ of the inhibitor solution in DMSO (or in the case of control

2.5 μΐ DMSO) and 15 μΐ enzyme solution (or enzyme dilution buffer in the case of background testing). The microtiter plates with the reaction mixture were then incubated for 30 minutes at 37 ° C and then quenched by the addition of 100 μΐ Biomol reagent. After 5 minutes, measure the absorbance at 650 nm.

Residual Activity Measurement (RA)

The residual activity of an enzyme is the ratio of the catalytic activities of an enzyme to an inhibitor and no inhibitor. The lower the RA value, the greater the inhibitor slows down the enzyme reaction at a given concentration. It is measured in two repetitions and calculated according to the following equation:

RA (%) = (Ai - A _with glue) / (AK-A _with glue) * 1 00

Ai = absorbance (λ = 650 nm) of the reaction mixture with enzyme and inhibitor

Asiepai ⁼ background absorbance of the reaction mixture without enzyme and with inhibitor

Ak ⁼ absorbance of the reaction mixture with enzyme and no inhibitor

AsiepaK ⁼ background of the reaction mixture without enzyme and without inhibitor

Determination of IC50

IC50 tells us the concentration of inhibitor needed to reduce the rate of MurD catalyzed reaction in half. It is determined at 700 μΜ D-Ala concentration and 500 μΜ ATP concentration. It is determined by measuring the RA of the DdlB enzyme at at least 7 different inhibitor concentrations. For each RA, we calculate the logit RA (logit RA = ln (RA / (1-RA)), and for each inhibitor concentration, expressed in μΜ, we calculate its decimal logarithm. I) + n If logit RA is zero then log c (I) is log IC50 IC50 is calculated by the formula: IC50 = 10 ^{n / k} .

II. Determination of bacterial susceptibility to potential antimicrobial compounds

To determine the minimum inhibitory concentration (MIC) of potential antimicrobial agents, the bacteria were cultured and diluted in IsoSensitest medium (Oxoid, Basingstoke, UK) using 10 ⁴ cells / ml Escherichia coli and 10 ⁶ cells / ml Staphylococcus aureus. Potential antimicrobial compounds were prepared in two concentrations in 50% DMSO (SigmaAldrich, Dorset, UK). The test compound and bacterial suspension were applied to 96-well microtiter plates (Nune, Fisher Scientific, Loughborough, UK) and then incubated for 16 hours in a Spectramax 384 plus microtiter reader (Molecular Devices, Abingdon, UK) using SOFTmax PRO 3.1.1. Optical density measurements were taken every 600 min at 600 nm. The plates were shaken for 30 seconds before each measurement. MIC values are concentrations of inhibitors that completely prevent bacterial growth.

The invention is explained, but in no way limited by the following embodiments:

EXAMPLE 1

Synthesis of A ^1- (4- / z <7-propylphenyl) -A ² - (2-chloroethyl) -1,2-diazendicarboxamide (H ₃ C) ₂ CH

NHC

N = N

CNHCH ₂ CH ₂ CI

Oh

Step 1

Methyl (4-isopropylanilino) carbonylhydrazincarboxylate

To a solution of triphosgene (1.10 g, 3.7 mmol) in dichloromethane (25 ml) and under argon, stirring vigorously at room temperature, a solution of 4-isopropylaniline (1.45 ml, 10 mmol) in dichloromethane (25 ml) was added dropwise. The resulting suspension was placed on an ice bath and triethylamine (3 ml, 23.3 mmol) was added. After 5 min, methyl hydrazincarboxylate (900 mg, 10 mmol) was added. The resulting yellowish solution was stirred for 10 min at 0 ° C and for 10 min at room temperature. HCl (30 ml, IM) was added and the white precipitated precipitate was removed, washed with water (10 ml) and cold diethyl ether (15 ml) and dried overnight. 2.01 g (80%) of the product are obtained.

T _tal . = 169.5-172.3 ° C (ethyl acetate)

IR (KBr) 3357, 3306, 3251, 2959, 1737, 1705, 1686, 1610, 1548, 1517, 1244 cm ^-1 .

1 H NMR (DMSO-δ / _d ) δ 1.17 (d, J = 6.8 Hz, 6H), 2.81 (h, J = 6.8 Hz, 1H), 3.60 (s, 3H), 7.11 (m, 2H), 7.35 (m, 2H), 7.95 (m s, 1H), 8.60 (s, 1H), 8.93 (m s, 1H).

¹³ C NMR (DMSO-d 6) δ 24.0, 32.7, 51.8, 118.6, 126.2, 137.3, 141.8, 155.6, 157.3.

MS (FAB) m / z (%) 252 (M ⁺ + H, 70), 91 (100).

Elem. Anal .: calculated for C _{I2 I7} H N ₃ O ₃ (M = 251.28 g / mol): 57.36% C, 6.82% H, 16.72% N; found: 57.15% C, 7.02% H, 17.01% N.

Step 2

Methyl (4-isopropylanilino) carbonyldiazenecarboxylate

To a suspension of methyl (4-isopropylanilino) carbonylhydrazincarboxylate (1.76 g, 7 mmol) in dichloromethane (20 ml) was added pyridine (1.13 ml, 14 mmol) with vigorous stirring at room temperature. During the slow addition of NBS (1.37 g, 7.7 mmol), the suspension was converted to a red solution, which, after stirring at room temperature for 1 h, was added an aqueous HCl solution (1: 1, 10 ml). The phases were separated and washed organically with 5% aqueous Na ₂ S ₂ O ₃ x 5H ₂ O solution (15 ml), saturated aqueous NaHCO ₃ solution (15 ml) and water (15 ml). The organic phase was dried with anhydrous Na ₂ SO4, the solvent was evaporated to give 1.70 g (98%) of product.

Ttai. ⁼ oil at room temperature

IR (NaCl) 3271,2961, 1773, 1732, 1602, 1559, 1519, 1437, 1417, 1251 cm ^-1 .

1 H NMR (DMSO-7 _rt ) δ 1.20 (d, 7 = 7.2 Hz, 6H), (h, 7 = 7.2 Hz, 1H), 4.07 (s, 3H), 7.29 (m, 2H), 7.64 (m ,

2H), 11.45 (s, 1H).

¹³ C NMR _(DMSO-fi 7) δ 23.8, 32.9, 55.6, 119.7, 126.8, 134.9, 145.4, 156.9, 161.9.

MS (FAB) m / z (%) 250 (M ⁺ + H, 53), 154 (61), 91 (68), 69 (80), 55 (100).

Elemental composition: Ci ₂ Hi ₅ N ₃ O _:

M = 249.27 g / mol

Step 3 / V ^1- (4-pheo-propylphenyl) - / V ² - (2-chloroethyl) -1,2-diazendicarboxamide

To a suspension of methyl (4- / isopropylanilino) carbonyldiazenecarboxylate (1.16 g, 4.65 mmol) and Na ₂ CO ₃ (477 mg, 4.5 mmol) in dichloromethane (7 ml) 2-chloroethylamine hydrochloride (522 mg, 4.5 mmol) was added portionwise. , stirring vigorously at 0 ° C. After stirring at 0 ° C for 2 h, the solvent was evaporated and the residue was suspended in ethyl acetate (15 ml). Shake the organic phase with water (2 × 10 ml), separate the phases, and dry the organic phase with anhydrous Na ₂ SO ₄ and evaporate the solvent. 1.09 g (82%) of the product are obtained.

T _ta i. = 148.5-150.4 ° C (dichloromethane)

IR (KBr) 3245, 2962, 1737, 1709, 1536, 1415, 1255, 1062, 830 cm ^-1 .

@ 1 H NMR (DMSO-7,) δ 1.20 (d, 7 = 7.1 Hz, 6H), 2.88 (h, 7 = 7.1 Hz, 1H), 3.63 (m, 2H), 3.78 (t, 7 = 5.8 Hz. 2H), 7.28 (m, 2H), 7.64 (m, 2H), 9.23 (broad t, 7 = 5.6 Hz, 1H), 11.27 (s, 1H).

¹³ C NMR (DMSO-7J δ 23.8, 32.9, 42.1,42.9, 119.7, 126.8, 135.2, 145.1, 158.1, 161.9.

MS (FAB) m / z (%) 299 ((M + 2) ⁺ + H, 47), 194 (85), 138 (100), 120 (61).

Elem. anal .: calculated for Ci ₃ H | ₇ C1N ₄ O ₂ (M = 296.75 g / mol): 52.62% C, 5.77% H, 18.88% N; found: 52.60% C, 5.97% H, 18.64% N.

EXAMPLE 2

Synthesis of / V ^L - (4-.vi?/£-butilfeniI) -N ² - (2-kIoroetil) -l, 2-diazendikarboksamida CH- V H ₃ CH ₂ CCH NHC

N = N cnhch ₂ ch ₂ ci

Step 1 π

Methyl (4-N- butylanilino) carbonylhydrazincarboxylate

To a solution of triphosgene (1.10 g, 3.7 mmol) in dichloromethane (10 ml) and under argon, stirring vigorously at room temperature, a solution of 4-secbutylaniline (1.53 ml, 10 mmol) in dichloromethane (10 ml) was added dropwise. The resulting suspension was placed on an ice bath and triethylamine (3 ml, 23.3 mmol) was added. After 5 min, methyl hydrazincarboxylate (900 mg, 10 mmol) was added. The resulting yellowish solution was stirred for 30 min at 0 ° C and 30 min at room temperature. HCl (15 ml, IM) was added and the white precipitated precipitate was removed, washed with water (7 ml) and cold diethyl ether (2x5 ml) and dried overnight. 2.14 g (81%) of the product are obtained.

T _tal . = 184-185.7 ° C (ethanol)

IR (KBr) 3362, 3308, 3250, 2959, 1736, 1707, 1686, 1609, 1549, 1248 cm ^1st

1 H NMR (DMSO-d / ₆ ) δ 0.76 (t, J = 7.3 Hz, 3H), 1.16 (d, J = 7.0 Hz, 3H), 1.51 (qd, J, = 7.3 Hz, J ₂ = 1.9 Hz , 2H), 2.51 (qt, J ₃ = 7.0 Hz, J ₂ = 1.9 Hz, 1H), 7.07 (m, 2H), 7.38 (m, 2H), 7.97 (wid s, 1H), 8.61 (s. 1H), 8.95 (broad s, 1H).

^I3 C NMR (DMSO-d / d) δ 12.0,21.8, 30.6, 45.5, 51.8, 118.6, 126.8, 137.4, 140.5, 155.6, 157.3. MS (EI) m / z (%) 265 (M ⁺ , 20), 175 (40), 146 (100), 90 (95).

HRMS; calculated for C ₁₃ Hi9N ₃ O ₃ : 265.1426; found: 265.1433.

Elem. anal .: calculated for C ₁₃ Hi9 N ₃ O ₃ (M = 265.31 g / mol); 58.85% C, 7.22% H, 15.84% N; found; 58.72% C, 7.42% H, 15.84% N.

Step 2

Methyl (4-seA-butylanilino) carbonyldiazenecarboxylate

To a suspension of methyl (4-sec-butylanilino) carbonylhydrazinecarboxylate (1.33 g, 5 mmol) in dichloromethane (15 ml) was added pyridine (810 pl, 10 mmol) under vigorous stirring at room temperature. During the slow addition of NBS (979 mg, 5.5 mmol), the suspension was converted to a red solution which, after stirring at room temperature for 1 h, added aqueous HCl (1: 1, 10 ml). The phases were separated and washed organically with 5% aqueous Na ₂ S ₂ O ₃ χ 5H ₂ O (15 ml), saturated aqueous NaHCO ₃ solution (7 ml) and water (10 ml). The organic phase was dried with anhydrous Na ₂ SO4, the solvent was evaporated to give 1.01 g (77%) of the oily product. T _ta i. ⁼ oil at room temperature

IR (NaCl) 3274, 2961, 1776, 1736, 1730, 1602, 1560, 1516, 1437, 1417, 1251 cm ^-1 .

1 H NMR (DMSO-ί / β) δ 0.77 (t, J = 7.3 Hz, 3H), 1.18 (d, J = 7.0 Hz, 3H), 1.55 (m, 2H), 2.60 (m, 1H), 4.07 (s, 3H), 7.25 (m, 2H), 7.64 (m, 2H), 11.45 (s, 1H).

¹³ C NMR (DMSO-? 4) δ 12.1, 21.7, 31.1,41.2, 55.4, 119.7, 127.9, 133.6, 145.8, 155.0, 162.1.

MS (FAB) m / z (%) 264 (M ⁺ + H, 41), 176 (100), 148 (85), 92 (65).

Elemental composition: C ₁₃ Hi ₇ N ₃ O ₃ M = 263.29 g / mol

Step 3

A ^1- (4-seA-butylphenyl) - N ^2- (2-chloroethyl) -1,2-diazendicarboxamide

To a suspension of methyl (4-seA-butylanilino) carbonyldiazenecarboxylate (1.00 g, 3.8 mmol) and Na ₂ CO ₃ (403 mg, 3.8 mmol) in ethyl acetate (15 ml), 2-chloroethylamine hydrochloride (441 mg, 3.8 mmol) was added portionwise. , stirring vigorously at 0 ° C. After stirring at 0 ° C for 4 h, the reaction was quenched by the addition of water (5 ml). Shake vigorously and separate the phase and dry organically with anhydrous Na ₂ SO ₄ and evaporate the solvent. 1.11 g (94%) of the product are obtained.

T _ta i. = 115.8-119 ° C (petroleum ether / ethyl acetate)

IR (KBr) 3246, 2965, 2928, 1735, 1708, 1601, 1532, 1416, 1317, 1308, 1247, 1190, 1059 cm ^-1 .

1 H NMR (DMSO-7,) δ 0.77 (t, J = 7.3 Hz, 3H), 1.18 (d, J = 7.0 Hz, 3H), 1.55 (qd, J, = J ₂ = 7.2 Hz, 2H). 2.59 (m, 1H), 3.63 (m, 2H), 3.78 (t, 7 = 5.9 Hz, 2H), 7.24 (m, 2H), 7.64 (m, 2H), 9.23 (mt, J = 5.5 Hz) , 1H), 11.27 (s, 1H).

¹³ C NMR (DMSO - </ ₆ ) δ 12.0, 21.7, 30.5, 40.4, 42.1, 42.9, 119.6, 127.4, 135.2, 143.9, 158.1, 161.9.

MS (FAB) m / z (%) 313 ((M + 2) ⁺ + H, 3), 185 (97), 93 (100).

Elem. anal .: calculated for C14H19CIN4O2 (M = 310.78 g / mol): 54.11% C, 6.16% H, 18.03% N; found: 54.30% C, 5.96% H, 17.91% N.

EXAMPLE 3

Synthesis of N - (4 - (tert -butylphenyl) - N ² - (2-chloroethyl) -1,2-diazenedicarboxamide cnhch ₂ ck ₂ ci

Step 1

Methyl (4-tert-butylanilino) carbonylhydrazincarboxylate

To a solution of triphosgene (549 mg, 1.85 mmol) in dichloromethane (10 ml) and under argon atmosphere, stirring vigorously at room temperature, a solution of 4-tertbutylaniline (800 μΐ, 5 mmol) in dichloromethane (10 ml) was added dropwise. The resulting suspension was placed on an ice bath and triethylamine (1.5 ml, 11.7 mmol) was added. After 5 min, methyl hydrazincarboxylate (450 mg, 5 mmol) was added. The resulting yellowish solution was stirred for 30 min at 0 ° C and 30 min at room temperature. HCl (20 ml, IM) was added and the white precipitated precipitate was removed, washed with water (7 ml) and cold diethyl ether (3x5 ml), and dried overnight. 1.20 g (91%) of the product are obtained.

T _mp = 185-187 ° C (dichloromethane / methanol)

IR (KBr) 3304, 2959, 1731, 1686, 1609, 1553, 1314,1259 cm ^-1 .

&Lt; 1 &gt; H NMR (DMSO- _dtf ) δ 1.25 (s, 9H), 3.61 (s, 3H), 7.25 (m, 2H), 7.37 (m, 2H), 7.96 (wid s, 1H), 8.62 (s , 1H), 8.94 (dec. S, 1H).

¹³ C NMR (DMSO-1L) δ 31.2, 33.8, 51.9, 118.3, 125.1, 137.0, 144.1, 155.6, 157.4.

MS (EI) m / z (%) 265 (M ⁺ , 20), 175 (32), 160 (100), 90 (95).

HRMS calcd for C13H19N3O3: 265.1426; found: 265.1431.

Elem. anal .: calculated for C13H19N3O3 (M = 265.31 g / mol): 58.85% C, 7.22% H, 15.84% N; found: 59.13% C, 7.47% H, 16.03% N.

Step 2

Methyl (4- / tert-butylanilino) carbonyldiazenecarboxylate

To a suspension of methyl (4-tert-butylanilino) carbonylhydrazincarboxylate (1.06 g, 4 mmol) in dichloromethane (10 ml) was added pyridine (645 μΐ, 8 mmol) with vigorous stirring at room temperature. During the slow addition of NBS (783 mg, 4.4 mmol), the suspension was converted to a red solution, which was added aqueous HCl (1: 1, 10 ml) after stirring at room temperature for 30 min. The phases were separated and washed organically with 5% aqueous Na ₂ S ₂ O3 χ 5H ₂ O (3 ml), saturated aqueous NaHCCh (10 ml) and water (10 ml). The organic phase was dried with anhydrous Na ₂ SO ₄ , the solvent was evaporated to give 963 mg (92%) of the oily product.

T _t ai. ⁼ oil at room temperature

IR (NaCl) 3270, 2960, 1773, 1730, 1601, 1560, 1522, 1437, 1406, 1252 cm ^-1 .

1 H NMR (DMSO-γ / β) δ 1.28 (s, 9H), 4.07 (s, 3H), 7.44 (m, 2H), 7.65 (m, 2H), 11.45 (s, 1H). ^{, 3} C NMR (DMSO-i / ₆ ) δ 31.0, 34.1,55.6, 119.4, 125.8, 134.6, 147.6, 157.0, 161.8. Elemental composition: Ci ₃ H | ₇ N ₃ O ₃ M = 263.29 g / mol

Step 3 y ^I - _(4-i> z-butylphenyl) ^{-N- 2} - (2-chloroethyl) -l, 2-diazendikarboksamid

To a suspension of 2-chloroethylamine hydrochloride (406 mg, 3.5 mmol) was added portionwise to suspensions of methyl (4- [ert-butylanilino) carbonyldiazenecarboxylate (920.5 mg, 3.5 mmol) and Na ₂ CO ₃ (371 mg, 3.5 mmol) in dichloromethane (5 ml). , stirring vigorously at 0 ° C. After stirring at 0 ° C for 2 h, the solvent was evaporated and the residue was suspended in ethyl acetate (15 ml). Shake the organic phase with water (2 x 10 ml), separate the phases and dry the organic phase with anhydrous Na ₂ SO ₄ . Evaporate the solvent to give 980 mg (90%) of the product.

T _W | = 125.9-127.7 ° C (ethyl acetate / petroleum ether)

IR (KBr) 3236, 3040, 2962, 1735, 1699, 1536, 1518, 1365, 1267, 835 cm ^-1 .

&Lt; 1 &gt; H NMR (DMSO-d / _tf ) δ 1.28 (s, 9H), 3.63 (m, 2H), 3.78 (t, J = 5.9 Hz, 2H), 7.43 (m, 2H), 7.64 (m, 2H) , 9.23 (broad t, J = 5.6 Hz, 1H).

¹³ C NMR (DMSO -? / ₆ ) δ 31.1, 34.1, 42.1, 42.9, 119.4, 125.7, 134.9, 147.4, 158.1, 161.9.

MS (FAB) m / z (%) 313 ((M + 2) ⁺ + H, 23).

Elem. Anal .: calculated for C _{) 4} H ₁₉ C ₁ N ₄ O ₂ (M = 310.78 g / mol); 54.11% C, 6.16% H, 18.03% N; found: 54.39% C, 6.42% H, 17.97% N.

EXAMPLE 4

Synthesis of / V ¹ - (2-kIoroetil) -N ² - (2-pyridylmethyl) -l, 2-diazendikarboksamida

cnhch ₂ ch ₂ ci o

To a solution of 2-picolylamine (155 pl, 1.5 mmol) in acetonitrile (3 ml) was added portionwise methyl (2chloroethylamino) carbonyldiazenecarboxylate (290.3 mg, 1.5 mmol; prepared according to Bombek, S.; Pozgan, F.; Kočevar, M.; Polanc , S., J. Org. Chem. 2004, 69, 2224-2227.), With vigorous stirring at O ° C. After stirring at 0 ° C for 45 min, the separated orange precipitate was removed and washed with cold acetonitrile (1 ml). 337.9 mg (83%) of the product is obtained.

T _that i = 142.6-144.1 ° C (ethyl acetate / methanol)

IR (KBr) 3247, 1746, 1712, 1594, 1537, 1474, 1437, 1360, 1265, 1225, 1192, 1063 cm ^-1 .

1 H NMR (DMSO-d, δ) δ 3.60 (m, 2H), 3.77 (t, J = 5.9 Hz, 2H), 4.55 (d, 7 = 6.0 Hz, 2H), 7.30 (ddd, J, = 7.5 Hz, J ₂ = 4.9 Hz, J ₃ = 1.1 Hz, 1H), 7.38 (ddd, 7; = 7.7 Hz, Λ = 1.1 Hz, J ₃ = 0.9 Hz, 1H), 7.80 (ddd, J, = 7.7 Hz , J ₂ = 7.5 Hz, J ₃ = 1.9 Hz, 1H), 8.54 (ddd, J, = 4.9 Hz, J ₂ = 1.9 Hz, J ₃ = 0.9 Hz, 1H), 9.09 (dil t, J = 5.7 Hz, 1H), 9.41 (broad t, J = 6.0 Hz, 1H).

¹³ C NMR (DMSO-d _d ) δ 42.0, 43.0, 45.3, 121.3, 122.5, 136.9, 149.1, 157.1, 161.8, 162.0.

MS (FAB) m / z (%) 270 (M ⁺ + H, 43), 154 (51), 135 (100).

Elem. anal .: calculated for C10H12CIN5O2 (M = 269.69 g / mol): 44.54% C, 4.48% H, 25.97% N; found: 44.43% C, 4.53% H, 26.03% N.

EXAMPLE 5

Synthesis of N-phenyl-7V '- (3-picolyl) diazendicarboxamide ao ch ₂ nhc

N = N

CNHPh n

Oh

To a solution of 3-picolylamine (0.610 ml, 6 mmol) in acetonitrile (3 ml) was stirred, stirring at 0 ° C, ethyl phenylaminocarbonyldiazenecarboxamide (1.327 g, 6 mmol; prepared according to Knight, GT; Loadman, JR; Saville, B .; Wildgoose, J., J. Chem. Soc., Chem. Commun. 1974, 193-194.). The resulting dense yellow precipitate was filtered off after 15 min, washed with acetonitrile to obtain 1.472 g (87%) of the product.

T _ta) . = 156-157 ° C (ethyl acetate).

IR (KBr) 3300, 2940, 1710, 1590, 1515, 1440, 1315, 1250, 1030 cm ^1st

1 H NMR (DMSO-7 ₆ ) 6 4.53 (d, 2H, 7 = 5.9 Hz), 7.19 (m, 1H), 7.41 (m, 3H), 7.72 (m, 2H), 7.77 (ddd, 1H, 7. = 7.8 Hz, 7 ₂ = 2.3 Hz, 7 ₃ = 1.6 Hz), 8.51 (dd, 1H, 7, = 4.8 Hz, J ₂ = 1.6 Hz), 8.59 (dd, 1H, 7, = 2.3 Hz, 7 ₂ = 0.8 Hz), 9.57 (t, 1H, 7 = 5.9 Hz), 11.31 (s, 1H).

¹³ C NMR (DMSO-7 ₆ ) δ 41.2, 119.6, 123.6, 124.9, 129.1, 133.6, 135.4, 137.4, 148.6, 148.9, 158.2, 161.9.

MS (FAB) m / z (%) 284 (M ⁺ + H, 30), 120 (14), 107 (25).

Elem. anal .; calculated for Ci4H ₁₃ N ₅ O ₂ (M = 283.29 g / mol): 59.36% C, 4.63% H, 24.72% N; found: 59.64% C, 4.53% H, 24.88% N.

EXAMPLE 6

Synthesis of A- (3-chlorophenyl) -A '- (3-picolyl) diazendicarboxamide

Step 1

Ethyl (3-chlorophenyl) aminocarbonyldiazenecarboxylate

Suspensions of ethyl (3-chlorophenyl) aminocarbonylhydrazincarboxylate (3.865 g, 15 mmol; prepared according to Bollbuck, G.; Stroh, H. H. Bamikow, G., J. Pract. Chem. 1971, 313, ΊΤ3-ΊΎ1.) And pyridine (2.43 ml, 30 mmol) in dichloromethane (60 ml) under vigorous stirring, NBS (2.723 g, 15.3 mmol) was added portionwise at room temperature. A red solution is formed. After 45 min HCl (1: 1, 40 ml) was added, phase separated and the organic phase was washed with aqueous Na ₂ S ₂ O3 * 5H ₂ O (1.5 g, 60 ml), with saturated aqueous NaHCO ₃ solution (60 ml). and with water (60 ml). The organic phase was dried with anhydrous Na ₂ SO4, the solvent was evaporated to give 3.731 g (97%) of the product.

Ttai. = 49.5-50.5 ° C (cyclohexane / toluene).

IR (KBr) 3300, 3000, 1780, 1740, 1600, 1490, 1250, 1030 cm ^-1 .

1 H NMR (DMSO-J ₆ ) δ 1.38 (t, 3H, J = 7.2 Hz), 4.53 (q, 2H, J = 7.2 Hz), 7.29 (ddd, 1H, 7, = 8.0 Hz, J ₂ = 2.1 Hz, 7 = 1.0 Hz), 7.46 (dd, 1H, 7 = 8.0 Hz, J ₂ = 8.2 Hz), 7.66 (ddd, 1H, 7 = 8.2 Hz, J ₂ = 2.0 Hz, 7 = 1.0 Hz), 7.85 (dd, 1H, 7 = 2.1 Hz, 7 = 2.0 Hz), 11.71 (s, 1H).

¹³ C NMR (DMSO-7) δ 13.9, 65.5, 111.2, 119.2, 124.9, 130.8, 133.4, 138.7, 157.0, 161.2.

MS (FAB) m / z 256 (M ⁺ + H, 16), 167 (9), 107 (25).

Elem. anal .: calculated for C10H10ClN3O3 (M = 255.66 g / mol): 46.98% C, 3.94% H, 16.44% N;

found: 47.20% C, 3.97% H, 16.36% N.

Step 2 / V- (3-Chlorophenyl) -7V '- (3-picolyl) diazendicarboxamide

To a solution of 3-picolylamine (0.610 ml, 6 mmol) in acetonitrile (2 ml) was stirred at 0 ° C, ethyl (3-chlorophenyl) aminocarbonyldiazenecarboxamide (1.534 g, 6 mmol) was added portionwise. The resulting dense yellow precipitate was filtered off after 15 min, washed with acetonitrile to give 1.412 g (74%) of the product.

T _la |. = 129-130 ° C (ethyl acetate).

IR (KBr) 3338, 2939, 1741, 1713, 1546, 1504, 1428, 1189 cm ^-1 .

1 H NMR (DMSO-7) δ 4.54 (d, 2H, J = 6.0 Hz), 7.27 (ddd, 1H, 7 = 8.0 Hz, J ₂ = 2.1 Hz, J ₃ = 1.0 Hz), 7.41 (ddd, 1H. 7 = 7.8 Hz, J ₂ = 4.8 Hz, 7 = 0.9 Hz), 7.45 (dd, 1H, 7 = 8.2 Hz, J ₂ = 8.0 Hz), 7.66 (ddd, 1H, 7 = 8.2 Hz, J ₂ = 2.0 Hz, Λ = 1.0 Hz), 7.78 (ddd, 1H, 7 = 7.8 Hz, J ₂ = 2.4 Hz, J ₃ = 1.7 Hz), 7.86 (dd, 1H, 7 = 2.1 Hz, 7 = 2.0 Hz), 8.52 (dd, 1H, 7, = 4.8 Hz, J ₂ = 1.7 Hz), 8.60 (dd, 1H, 7 = 2.4 Hz, J ₂ = 0.9 Hz), 9.63 (t, 1H, J = 6.0 Hz), 11.55 ( s, 1H).

^I3 C NMR (DMSO-7) δ 41.2, 118.1, 119.0, 123.6, 124.7, 130.9, 133.4, 133.6, 135.4, 138.9, 148.6, 148.9, 158.2, 161.7.

MS (FAB) m / z (%) 318 (M ⁺ + H, 30), 123 (22), 107 (33).

Elem. anal .: calculated for C14H12CIN5O2 (M = 317.73 g / mol): 52.92% C, 3.81% H, 22.04% N; found: 53.14% C, 3.88% H, 21.72% N.

EXAMPLE 7

Results of biological tests to determine the efficacy of Ddl inhibitors:

Structure Ddl MIC E.coli 1411 E.coli SM 1411 S. aureus 8325-4 N H> 1 Y 0 IC ₅₀ = 119pM > 256 > 256 > 256 p ⁿ == n / ° / = C V7 IC _S0 = 158pM > 256 > 256 > 256 0 IC _S0 = 49pM > 256 > 256 > 256 ^H Ϊ 0 IC _S0 = 25pM > 256 > 256 > 256 χ-γ \ _κ Χζ ^Ν γ ^κ \ ^ Ο IC ₅₀ = 123pM > 256 > 256 > 256 α o IC ₅₀ = 187pM > 256 > 256 > 256 0 ^c, \ / K. ψ Cl 0 IC ₅₀ = 133pM > 256 > 256 > 256 0 α - 'r'O HBF4 ^N ΙΟ ₅₀ = 76μΜ > 256 > 256 > 256

Structure Ddl MIC E.coli 1411 E.coli SM 1411 S.aureus 83254 CH, L ^ch - Ax _x · N H II ° Y 0 IC _S0 = 37pM > 256 > 256 > 256 Cu «y qV ΙΟ ₅₀ = 111μΜ 64 64 64 0 q ^ A-y ~ q Cl IC ₅ o = 15pM 64 64 256 0 IC50—3611M 64 64 128 CUV _Y yO IC50 ^with 41pM > 256 > 256 > 256

PATENT APPLICATIONS

Claims (6)

1. Compounds of general formula I, X ¹ OOX ² i! I II II I 9 9 W-Y- NC- N = NC- N-γ-W ² (I) in which: 1 Ύ X, X: hydrogen, Ci. ₆ linear or branched alkyl, (aryl) -NH-, -O-, - (CH 2) _n - (wherein n may be 0 to 5), C 1-6 branched alkyl; Υ ¹ , Y ² : - (CH ₂ ) _n - (wherein n may be 0 to 5), C 1-6 branched alkyl; -NH-, -C = O; -NHCO-, -C0NH-; W \ W ² : - hydrogen, - C1-6 linear or branched alkyl which may be substituted by one or more nitro groups, by one or more halogens, by trifluoromethyl group, by one or more hydroxy groups, - phenyl, which may be unsubstituted or substituted with one or more nitro groups, with one or more halogens, with trifluoromethyl group, with one or more hydroxy groups, with one or more C1-8 linear or branched alkyl, with one or more Ci -8 linear or branched alkoxy group, with one or more unsubstituted or substituted phenyl groups, - unsubstituted or substituted naphthyl, - fluorenyl, - a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic ring system, which may be substituted or unsubstituted and which may contain, in addition to carbon atoms, up to 3 heteroatoms selected from N, O, P and S, - a 3- to 10-membered monocyclic or 7- to 10-membered bicyclic carbocyclic ring system, which may be substituted or unsubstituted and which may contain, in addition to carbon atoms, up to 3 heteroatoms selected from N, O, P and S. Compounds of formula I according to claim 1 for use as therapeutically active substances. Process for the preparation of compounds of the formula I according to claim 1, characterized in that a) the asymmetric derivative is formed by reacting the isocyanate of formula II (prepared from the corresponding amine with phosgene, diphosgene or triphosgene in an aprotic dry solvent in the presence of a tertiary amine) with carbazate III (R = C 1-6 linear or branched alkyl) in an aprotic solvent, W ¹ - Y — N — C = O (Π) Oh H, NN— ² H OR (III) gives the corresponding 1,4-disubstituted semibarbazide IV; the same compound can be prepared from 4-substituted semicarbazide V if it is reacted with chloroformate VI (R = C _1-6 linear or branched alkyl); X ¹ OOX ¹ O. . I II II ₂ , 1 1 I II O W ¹ —Y — N — C — N — N — C — O — Y — Vv W — Υ ^ -Ν-Ο-Ν-ΝΚ II HHH ² Cl-U-OR (IV) (V) (VI) is formed with the appropriate organic or inorganic oxidant to form the 1,4-disubstituted semicarbazide IV with amino or carboxylic acid VII by the substitution of an alkoxy group with primary or secondary amine VIII to diazendicarboxamide I; X ¹ O Ν-Υ-VV ² H W ¹ - Y ¹ -NCN = NC-OR (VII) (Vlil) b) the symmetric derivative of diazendicarboxamide I (X ¹ = X ² , Υ ¹ = Y ² , W '= W ² ) is obtained if the dialkyl diazendicarboxylate is treated with at least two equivalents of primary or secondary amine VIII in a protic or aprotic solvent, or without the presence of a solvent, with the nucleophilic substitution of the two alkoxy groups. Use of compounds of general formula I according to claim 1 for the preparation of medicaments used for the inhibition of Ddl in bacterial infections. Pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula I according to claim 1 and pharmaceutically acceptable excipients. Pharmaceutical preparations according to claim 5, characterized in that they are used to inhibit Ddl in bacterial infections of humans and other mammals.